# Team:UCL/Project/Protocols

(Difference between revisions)
 Revision as of 09:49, 26 September 2013 (view source)← Older edit Revision as of 09:50, 26 September 2013 (view source)Newer edit → Line 98: Line 98:
-

Minimal Agar/p> +

Minimal Agar

Mix: Mix: Line 131: Line 131:

39 mL 39 mL
+ + + + +
+
+
+
+

1.4% Agar

+

+ In 500mL Duran bottle insert: +

+ +
ReagentRequired Quantity
Agar7g
RO H2O500mL
+ + + + + + + + + + + +

## Revision as of 09:50, 26 September 2013

There are two solutions to $$ax^2 + bx + c = 0$$ and they are $$x = {-b \pm \sqrt{b^2-4ac} \over 2a}.$$

Bacterial Lab Protocols

5X M9 Salts

In 500mL Duran bottle insert:

Reagent Required Quantity
Na2HPO4 32g
KH2PO4 7.5g
NaCl 1.25g
NH4Cl 2.5g
RO HCL 500ml

Minimal Agar

Mix:

Reagent Required Quantity
5x M9 Salts 10mL
2 mg/ml Thiamine 50µl
20% D Glucose 1 mL
1M CaCl2 5µl
1M MgSO4 100µl
1.4% Agar 39 mL

1.4% Agar

In 500mL Duran bottle insert:

Reagent Required Quantity
Agar 7g
RO H2O 500mL
expands with content

Mammalian Lab Protocols

For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.

1) The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.

2) Incubate cells in their normal growth conditions (37^0 C and 5% CO2)for 24 hours.

3) Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.

4) Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.

5) Incubate at room temperature for 5-10mins to allow for complex formation.

6) Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml.

7) Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well.

8) Change medium and wash with PBS.

9) Incubate for 24-48 hours.

9) Assay fro gene expression.